Eukaryotic cells are defined by the presence of membrane-delineated organelles, providing the necessary environments for various biochemical reactions. In plants, three organelles - chloroplasts, mitochondria and peroxisomes - are critically involved in many essential aspects of plant physiology, including energy capture, conversion, storage and metabolism. The major protein machineries governing the dynamics of these organelles, including those for protein import and membrane fission, have been identified. However, mechanisms that regulate the major machineries are just beginning to be elucidated in plants and other systems. This dissertation research aims to deepen the understanding of the roles of protein post-translational modifications and membrane lipids in regulating organelle dynamics using the plant model system Arabidopsis thaliana. First, I studied the role of cardiolipin (CL), a negatively charged non-bilayer forming phospholipid, in organelle dynamics and plant development. I showed that CL is important for mitochondrial fission in Arabidopsis and exerts this function, at least in part, through stabilizing the higher-order protein complex of dynamin-related protein 3 (DRP3), a major division protein for mitochondria and peroxisomes. CL and Cardiolipin Synthase (CLS) both localize specifically to mitochondria. CL deficiency resulted from CLS gene disruption or suppression of gene expression leads to mitochondrial elongation and enlargement, abnormal mitochondrial cristae, plant dwarfness and susceptibility to various abiotic stresses. Then I focused on a key form of protein post-translational modification - ubiquitination, for its role in regulating organelle dynamics. Through a bioinformatic approach, I identified a mitochondrial outer membrane associated deubiquitinase, UBP27, and determined its membrane topology, targeting signal and enzymatic activity. UBP27 is anchored to the mitochondrial outer membrane with the enzymatic domain facing the cytosol. Overexpression of UBP27 reduces mitochondrial length and mitochondrial association of DRP3, suggesting its role in mitochondrial dynamics possibly through affecting the recycle of DRP3 from mitochondrial to the cytosol. Finally, I identified a small family of RING domain-containing proteins, namely SP1, SPL1 and SPL2, among which ubiquitin E3 ligase activity had been shown for SP1. SP1 and SPL1 localize to chloroplasts, mitochondria and peroxisomes, while SPL2 localizes to chloroplasts and mitochondria; such proteins that localize to all three major energy organelles had not been reported. I performed initial characterization of these RING domain proteins, and discovered distinct characteristics among them in targeting signal, membrane association, and loss- and gain-of-function mutant phenotypes. I hypothesize that these three related proteins may coordinately regulate the import, division, and/or distribution of chloroplast, mitochondria and peroxisomes. In summary, my work has revealed multiple regulatory mechanisms in the dynamics of Arabidopsis mitochondria, peroxisomes and chloroplasts, key organelles that act in concert in a number of plant metabolic pathways essential for energy metabolism.
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